Summary Genomics technology is in the midst of yet another revolution, this time focusing on the analysis of long, intact genomic DNA molecules. These long-read technologies, which include nanopore sequencing, genome mapping in nanochannels, and droplet-based barcoding, aim to alleviate the short-read length problem in next- generation sequencing (NGS). Long read-length technologies, either in isolation or combined with NGS, represent a transformative breakthrough that addresses current limitations in genome sequencing, assembly, and analysis. As long-read technologies begin to mature, the bottleneck in their further advancement is moving to sample preparation steps. While tremendous innovations were required to develop long-read technologies, the methodology for obtaining the long DNA molecules that go into to these new devices has seen little innovation. The standard method for extracting genomic DNA is to embed the cells in an agarose plug and extract the DNA. This technology, developed over 30 years ago for chromosome sizing by pulsed-field gel electrophoresis, remains the state-of-the-art today with only minor incremental advances. This proposal provides a generic method that can be used to create a long DNA sample for any long-read technology. The innovation in our project is recognizing that microfluidics can substantially reduce the reactor volume for DNA extraction, and thus massively reduce the processing time, while maintaining the yield needed for genomics. In Specific Aim 1, we will develop a microfluidic system based that removes diffusive limitations in DNA extraction from cells, reducing the processing time by 100-fold. In Specific Aim 2, we will develop an electrophoretic method to recover the DNA, again reducing the time relative to the state-of-the-art method by more than an order of magnitude. In Specific Aim 3, we will combine optimal designs from the previous aims to demonstrate DNA recovery from a human cell line and show that these DNA are of sufficient quality for genome mapping in nanochannels. Taken together, the innovative features of our platform will reduce genomic DNA extraction from a labor intensive, day-long protocol to an automated, hour-long protocol. This project takes advantage of an innovative academia/industrial collaboration between the University of Minnesota and BioNano Genomics (BNG), established over the past three years, that leverages the unique capabilities of both teams. The microfluidic device design will take place at Minnesota, where there is expertise for device fabrication and prototyping. The testing of the device in a real-world environment will take place at BNG, where there is expertise in genome mapping in nanochannels for human cells.